Extracellular matrix (ECM) proteins have an important function in providing structural integrity to tissues and in presenting proper environmental cues for cell adhesion, migration, growth, and differentiation. All of these aspects rely on the spatiotemporal expression of adhesive as well as anti-adhesive components in extracellular matrices and on the cell surface. These include “classical” ECM proteins like fibronectin and laminin that act as anti adhesive ligands under certain experimental conditions. Many proteins that inhibit cell adhesion were classified as matricellular proteins. This group embodies proteins such as thrombospondin, tenascin, and hevin that do not serve as integral components of matrix elements but rather function through binding to matrix proteins as well as to cell surface receptors. Cell-matrix interactions depend to a large extent upon the engagement of specific ECM proteins with cell surface integrins (Hynes R O 1999 Trends Cell Biol 9:M33–M37; Giancotti F G & Ruoslahti E 1999 Science 285:1028–1032). Cell adhesion also depends upon carbohydrate-protein interactions, mediated by mammalian lectins of different families (Sharon N & Lis H 1995 Essays Biochem 30:59–75).
Lectins are proteins that are defined by their ability to bind carbohydrates specifically and to agglutinate cells. Lectins have been shown to be involved in a wide variety of cellular functions including cell-cell and cell-matrix interactions. Lectins are widespread among plants, invertebrates and mammals.
Animal lectins have been grouped into four distinct families: C-type lectins, which include selectins; P-type lectins; galectins (formerly termed S-type lectins or S-Lac lectins); and pentraxins (Barondes S H et al. 1994 J Biol Chem 269:20807–20810).
Galectins require fulfillment of two criteria: affinity for β-galactosides and significant sequence similarity in the carbohydrate recognition domain (CRD) (Hirabayashi J & Kasai K 1991 J Biol Chem 266:23648–23653). Galectin-1 and -2 are homodimers with subunit molecular weight of ˜14 kDa, that are not subjected to post-translational modifications (Tracey B M et al. 1992 J Biol Chem 267:10342–10347). Galectin-1 and galectin-3 are the best characterized of the mammalian galectins. Galectin-1 is found in the extracellular matrix aid has been shown to interact with laminin. It is known to both promote and inhibit cell adhesion. In skeletal muscle, galectin-1 inhibits cell matrix interaction and is thought to play a role in muscle development while in other cell types galectin-1 promotes cell-matrix adhesion. Galectin-1 has also been implicated in the regulation of cell proliferation and in some immune functions. Over-expression of galectin-1 has been also shown to correlate with tumor metastasis potential (Raz A et al 1986 Cancer Res 46:3667–3672).
Galectin-3 is a monomeric protein, composed of an N-terminal half made of tandem repeats characteristic of the collagen superfamily, and a C-terminal half homologous to galectin-1 and -2. Like galectin-1, galectin-3 binds to laminin. It is known to plays a role in inflammation by binding to both IgE and IgE receptor thereby causing activation of mast cells and basophils. Galectin-3 has been shown to concentrate in the nucleus of certain cell types during proliferation. Expression of galectin-3 is elevated in certain tumors, suggesting that galectin-3 plays a role in metastasis. Indeed overexpression of galectin-3 in a weakly metastatic cell line caused a significant increase in metastatic potential (Raz A et al 1990 Int J Cancer 46:871–877).
Galectin-4 was cloned from rat intestine, and a homologous protein was cloned from nematode. Galectin-4 is a monomer with molecular mass of 36 kDa. It contains tandem domains of ˜140 amino acid each, homologous to galectin-1 and -2, that are separated by a link region. The function of galectin-4 is presently unknown.
The expression of human galectin-7 appears to be limited to keratinocytes. Galectin-7 is though to play a role in cell-matrix and cell-cell interactions as galectin-7 is sharply downregulated in anchorage independent keratinocytes and is absent in malignant keratinocytes cell lines. Galectin-7 may be required for the maintenance of normal keratinocytes (Madsen P et al J Biol Chem 270:5823–5829).
One of the present inventors has previously disclosed (U.S. Pat. No. 5,908,761) rat galectin-8, which is a 34-kDa secreted protein expressed in a wide variety of tissues in adult rats including lung, liver, kidney, spleen and cardiac and skeletal muscles. This disclosure is incorporated herein in its entirety by reference. Galectin-8 is composed of two homologous (38% identity) carbohydrate recognition domains joint by a short (˜26 amino acids) linking peptide. The link region of rat galectin-8 is not similar to either the link region of galectin-4 or to the proline, glycine and tyrosine-rich repeat domain of galectin-3.
Galectin-8 is a secreted protein, although lacking a signal peptide. Trypsinization experiments have shown that a significant fraction of the secreted galectin-8 remains bound to the extracellular surface. Galectin-8 form tight complexes with a selective subset of integrins, and secreted, soluble galectin-8 was shown to specifically inhibit cell adhesion. Over-expression of galectin-8 in 1299 cells, a cell line derived from human non-small cell lung carcinoma, significantly promoted inhibition of cell adhesion. However, full-length antisense galectin-8 showed no effect on the adhesion of these cells (Hadari Y R et al 2000 J Cell Sci 113:2385–2397).
Human galectin-8 has also been cloned (U.S. Pat. Nos. 5,869,289; 6,281,333), and was shown to share features with rat galectin-8 as well as other mammalian galectins, which are involved in the regulation of cell growth and development, including metastatic potential. WO99/43857 discloses that prostate carcinoma tumor antigen-1 (PCT-1), the human isoform of galectin-8, is highly expressed in certain forms of prostate carcinomas (Su Z-Z et al 1996 Proc Natl Acad Sci U.S.A. 93:7252–7257). Galectin-8 is over-expressed in lung carcinomas and is also overexpressed in the invasive regions of xenografted glioblastomas (Camby I et al 2001 Brain Pathol 11:12–26). In contrast, galectin-8 expression decreased in colon cancer compared with normal human colon tissue (Nagy et al 2002 Gut 50:392–401.
The diagnostic utility of human galectin-8 was proposed (U.S. Pat. No. 6,281,333), and PCT antigen-1 (WO99/43857) has been disclosed as useful for the detection of metastatic cancer cells. However, WO99/43857 fails to teach the differences in galectin-8 expression in various tumor types, and thus provides no guidance for distinguishing malignant cancers from benign hyperproliferative disorders, thus rendering the claimed diagnostic method unreliable.
Preliminary in vitro results of the present inventors and coworkers (Levy Y et al 2001 J Biol Chem 276:31285–31295) have shown galectin-8 as a physiological modulator of cell adhesion, capable of both promoting and inhibiting cell adhesion depending on its configuration.
Cell adhesion is critical to the development and survival of multicellular organisms. The process of cell adhesion is complex, requiring participation of extracellular proteins such as fibronectin, vitronectin, collagen, laminin and galectin and numerous families of cellular receptors such as the integrins and cellular adhesion molecules (CAMS). These molecules are involved in the adhesion of both normal and malignant cells, and therefore are crucial to neoplastic proliferation and metastasis.
In view of the accumulated information described above, ECM proteins and matricellular proteins such as galectins are assumed to play a role in modulating cell-matrix interactions in a variety of pathological processes, including tumor development. However, the therapeutic utility of galectins in general, and galectin-8 in particular, were never actually reduced to practice for treatment of cell-proliferative pathological disorders.
Thus, there is a recognized need for, and it would be highly advantageous to have methods of regulating expression or function of galectin-8 and galectin-8 like proteins and using same as a means for modulating cell interactions in vivo. More particularly, there is an unmet need for method of using these proteins as a means of inhibiting tumor cell growth.